Twenty-five percent of U.S. women report frequent use of vaginal lubricants to reduce discomfort and increase pleasure during sex. Clinically, lubricants are also used for digital exams and procedures such as trans-vaginal ultrasound (T-VUS). Among postmenopausal women, 50% use lubricants due to vaginal dryness and dyspareunia. The first line treatment for these women is non-hormonal vaginal lubricants. Recent studies have questioned the safety of current lubricants, yet there is little available data A healthy vagina is protected by Lactobacillus spp. that acidify the vagina with lactic acid, a potent bacteriocide and viricide. Disruption of this healthy microbiome markedly increases susceptibility to reproductive tract infections. We hypothesize that lubricants can disrupt the vaginal microbiome and increase risk to infections. Most vaginal lubricants contain hypertonic humectants such as glycerol or propylene glycol together with antibacterial preservatives, parabens and sometimes chlorhexidine. The active ingredients are generally recognized as safe (GRAS) by the FDA, hence are not subject to human clinical trials. However, at high concentrations or with frequent use, these ingredients may have toxic effects. It is unknown if hypertonic antibacterial lubricants affect the vaginal microbiota. We propose three aims to study the effect of lubricant on the vaginal microenvironment: 1) Evaluate the effects of lubricant on the temporal dynamics of vaginal microbiota with two methods: 1A) Perform a controlled observational study of one-time use of vaginal lubricant by recruiting women who are presenting for T-VUS, a procedure in which lubricant is applied to a T-VUS probe. This study will standardize the lubricant brand and dose by using the hypertonic lubricant currently in use by the Department of Radiology. Participants will self-collect daily samples prior to T-VUS for 1 week and twice-weekly for 9- weeks after TVUS. Changes in bacterial composition will be assessed using 16S rRNA gene analysis. 1B) Conduct a secondary analysis of existing data (behavioral diaries, clinical exams and 16S rRNA sequences) collected from a cohort of 130 women who were followed for 10 weeks in a prior study as part of the Human Microbiome Project (HMP). Participants self-collected mid-vaginal swabs daily and all samples are stored in a freezer repository. Lubricant use was reported by 37 women on 140 occasions. Samples collected every other day have already undergone DNA extraction and 16S rRNA sequencing (NIH RO1-GM103604). 2) Examine the effects of lubricant on the vaginal microenvironment in samples obtained from Aims 1A and 1B by assessing pH, D- and L-isomers of lactic acid and inflammatory cytokines. 3) Determine if lubricants have toxic effects on the vaginal epithelium using measurements of cell shedding rates, glycogen (necessary for lactobacilli survival), fibronectin (a marker of epithelial disruption), and lactate dehydrogenase (a marker of cell death) in samples collected in Aim 1A. This study will contribute to the development of safer formulations for personal and clinical lubricants.